A novel chorismate mutase effector secreted from root-knot nematode Meloidogyne enterolobii manipulates plant immunity to promote parasitism

Meloidogyne spp.is an economically important plant-parasitic nematode distributed worldwide.To fight with host immune system for successful parasitism,plant parasitic nematodes secrete effectors to promote infection.In this study,we identified one chorismate mutase(CM)effector from M.enterolobii,named Me-CM.Spatial and temporal expression assays exhibited Me-cm is expressed in esophageal glands and up-regulated at parasitic-stage juveniles.Me-CM affects the pathogenicity of M.enterolobii based on the reduced infection rate,number of galls,egg masses,eggs per mass and multiplication rate collected from RNA silencing experiments.We showed that Me-CM localized in the cytoplasm and nucleus of plant cells and decreased the expression level of the marker gene PR1 of salicylic acid(SA)pathway.Besides,constitutive expression of Me-cm in Arabidopsis thaliana significantly reduced salicylic acid concentration.These results suggested that M.enterolobii may secrete effector Me-CM to fight with plantimmunesystemsvia regulating SA signaling pathway when interacting with host plants,ultimately facilitating parasitism.

A chorismate mutase from Radopholus similis plays an essential role in pathogenicity

In the process of infecting plants, plant parasitic nematodes release a series of proteins that play an essential role in the successful infection and pathogenesis of plant cells and tissues through stylets or body walls. In this study,based on transcriptome data, a chorismate mutase gene of Radopholus similis(RsCM) was identified and cloned,which is a single copy gene specifically expressed in the oesophageal gland and highly expressed in juveniles and females. Transient expression of RsCM in tobacco leaves showed that it was localised in the cytoplasm and nucleus of tobacco leaf cells, which inhibited the pattern-triggered immunity(PTI) induced by flg22, including callose deposition and defence gene expression, and cell death induced by immune elicitors BAX, but could not inhibit cell death induced by immune elicitors Gpa2/RBP-1. The RNA interference(RNAi) transgenic tomato of RsCM obviously inhibited the infection, pathogenicity, and reproduction of R. similis. However, the resistance of the overexpression transgenic tomato of RsCM to R. similis infection was significantly reduced, and the expression levels of two salicylic acid(SA) pathway genes(PR1 and PR5) in roots infected by the nematode were significantly down-regulated,which indicated that RsCM might be involved in the inhibition of SA pathway. The results of this study demonstrate that RsCM suppresses the host immune system and might be a new target for the control of R. similis, which also provides new data for the function and mechanism of CM genes of migratory parasitic plant nematodes.

Role of vitamin B_(12) on methylmalonyl-CoA mutase activity

Vitamin B 12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes,which have been grouped into three subfamilies depending on their cofactors.Among them,methylmalonyl-CoA mutase (MCM) has been extensively studied.This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate.The crystal structure of MCM determined in Propionibacterium freudenreichii var.shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme,coenzyme,and substrate.The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism.The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein,there are significant differences in the structural organization of the two proteins.Recent studies have identified the involvement of an accessory protein called MMAA,which interacts with MCM to prevent MCM's inactivation or acts as a chaperone to promote regeneration of inactivated enzyme.The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase,the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans.It is still necessary to study the mechanisms involved in more detail using new methods.

Clinical and prognostic significance of expression of phosphoglycerate mutase family member 5 and Parkin in advanced colorectal cancer

BACKGROUND Drugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer(CRC)cells.Phosphoglycerate mutase family member 5(PGAM5)activates serine/threonine PTEN-induced putative kinase 1/Parkin pathway-mediated mitophagy.However,there are few studies on the clinical and prognostic significance of expression of PGAM5 protein and mitophagy-related protein Parkin in patients.AIM To assess the clinical significance of PGAM5 and Parkin proteins,as biomarkers for diagnosis and prognosis of CRC,by studying their expression in advanced CRC tissues and their association with clinicopathological parameters.METHODS The expression of PGAM5 and Parkin in CRC tissues from 100 patients was determined by immunohistochemistry.Each case was evaluated by using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-4).The final staining score was calculated as the intensity score multiplied by the proportion score.Specimens were categorized as either high or low expression according to the Youden index,and the association between the expression of PGAM5 or Parkin and clinicopathological factors was ascertained.Additionally,we employed western blot to measure PGAM5 and Parkin protein expression in six matched pairs of CRC and adjacent non-tumor tissues.RESULTS Immunohistochemical and western blot findings showed that both PGAM5 and Parkin protein expression in tumor tissues was significantly higher than that in the adjacent tissues:PGAM5 and Parkin were mainly expressed in the cytoplasm of colonic epithelial cells.PGAM5 and Parkin protein levels were significantly positively correlated in advanced CRC tissues.Moreover,reduced Parkin protein expression was an independent prognostic factor for overall survival and progression-free survival in CRC patients as evinced by multivariate analysis.CONCLUSION The expression of PGAM5 protein and mitophagy-related protein Parkin has diagnostic significance for CRC and may become new biomarkers.Parkin may be a potential marker for the survival of CRC patients.

Kinetic Studies of a Coenzyme B12 Dependent Reaction Catalyzed by Glutamate Mutase from <i>Clostridium cochlearium</i>

The coenzyme B12 dependent glutamate mutase is composed of two apoenzyme proteins subunits;S and E2, which while either fused or separate assemble with coenzyme B12 to form an active holoenzyme (E2S2-B12) for catalyzing the reversible isomerization between (S)-glutamate and (2S, 3S)-3-methylas- partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2S, 3S)-3-methylaspartate to form mesaconate (λmax = 240 nm, Ɛ240 = 3.8 mM-1·cm-1). The activities of different reconstitutions of glutamate mutase from separate apoenzyme components S and E in varied amounts of coenzyme B12 and adenosylpeptide B12 as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis- Menten Method. The values of Km for coenzyme B12 in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as;1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B12;1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously associates the increasing affinity of cofactors to apoenzyme with increased amount of component S used in reconstituting holoenzyme from separate apoenzyme components and cofactor. Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (λmax = 340 nm, Ɛ340 = 6.3 mM-1·cm-1) as determined by UV spectrophotometry after addition of (2S, 3S)-3-methylaspartate and pyruvate to a mixture of E2S2-B12 and two auxiliary holoenzymes system;pyridoxal-5-phosphate dependent glutamate-pyruvate aminotransferase and NADH dependent (R)-2-hydroxyglutarate dehydrogenase. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (S)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (R)-2-hydroxylglutarate dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2S, 3S)-3-methylaspartate in the reaction of glutamate mutase as Km= 7 ± 0.07 mM and kcat= 0.54 ± 0.6 s-1. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, Keq = [(S)-glutamate] × [(2S, 3S)-3-methylaspartate]-1 = 16, where the kinetic constants of (S)-glutamate were determined by the standard methylaspartase coupled assay.

人参UDP-阿拉伯糖变位酶基因电子克隆及分析

UDP-阿拉伯糖变位酶是阿拉伯糖合成过程的关键酶,在植物形态建成和逆境胁迫响应过程中起着重要的作用.UDP-阿拉伯糖变位酶可使UDP-Araf和UDP-Arap相互转换,参与包括阿拉伯糖在内的多糖合成.以玉米UDP-阿拉伯糖变位酶氨基酸序列作为探针,通过电子克隆的方式,对人参EST数据库进行检索,获得具有高度同源性的cDNA序列,采用DNAMAN软件对其进行拼接和组装,最终获得一条长为1445 bp的cDNA序列.利用相关软件对人参UDP-阿拉伯糖变位酶基因进行生物信息学分析.结果表明:人参UDP-阿拉伯糖变位酶蛋白为稳定的亲水性蛋白,该蛋白上的磷酸化修饰有33处,无跨膜螺旋区,不存在信号肽,为非分泌蛋白.该蛋白含有无规则卷曲、α螺旋、延伸链、β折叠二级结构,其中无规则卷曲结构最多.人参UDP-阿拉伯糖变位酶的氨基酸序列与凤仙花同源性较高,与石榴花同源性较低.分析结果能为人参后续相关研究提供一定的参考依据.

G-四链体在糖酵解相关基因中的分布及调控

目的探讨G-四链体(G4)在糖酵解相关基因中的分布及调控。方法选取200个糖酵解相关基因转录起始位点上游1500 bp至5′非翻译区的序列进行生物信息学分析,初步确定含有潜在G-四链体形成序列(PQS)的相关基因;圆二色谱法和非变性聚丙烯酰胺凝胶电泳法检测G4形成;外切酶Ⅰ(ExoⅠ)水解实验在0,0.5,2,8,16和32 min检测G4稳定性;构建将相关基因的特定片段插入到荧光素酶表达序列之前的报告基因质粒,双荧光素酶报告基因系统检测荧光素酶的表达水平进而判断G4对启动子活性的影响;实时荧光定量PCR检测荧光素酶的mRNA水平进一步验证G4的转录调控作用。结果①生物信息学分析表明,200个糖酵解相关基因中有12个基因含有PQS,进一步结合PQS长度及结构分析,醛缩酶A(ALDOA)和磷酸变位酶2(PGAM2)的PQS理论上可形成稳定G4。②ALDOA和PGAM2均在260 nm处有最大正吸收峰,在240 nm处有最大负吸收峰,符合平行结构G4的特征构象,同时二者的PQS可形成G4。③ExoⅠ消化后,ALDOA和PGAM2无明显水解,证明G4具有稳定性,同时二者的PQS突变后均可逐渐被水解。④ALDOA突变后荧光素酶的表达水平和mRNA水平显著升高(P<0.01);PGAM2突变后荧光素酶的表达水平和mRNA水平显著降低(P<0.01)。结论糖酵解相关基因的序列中存在大量PQS,其中ALDOA和PGAM2的PQS可形成稳定G4,并具有转录调控作用。

CircOGDH通过调控miR-195-5p/PGAM5轴对氧糖剥夺/复糖复氧诱导的小胶质细胞损伤的影响

目的:探讨环状RNA OGDH(CircOGDH)通过调控miR-195-5p/磷酸甘油酸变位酶5(PGAM5)轴对氧糖剥夺/复糖复氧(OGD/R)诱导的小胶质(MG)细胞损伤的影响。方法:将体外培养的HMC3细胞分为:NC组(正常培养)、OGD/R、si-NC组、si-CircOGDH组、mimic NC组、miR-195-5p mimic组、si-CircOGDH+inhibitor NC组、si-CircOGDH+miR-195-5p inhibitor组。除NC组外,剩余组在转染对应物质前构建OGD/R模型。qRT-PCR法测定CircOGDH、miR-195-5p、PGAM5 mRNA表达水平;测定各组细胞增殖、凋亡、活性氧(ROS)及炎性因子水平;Western blot检测Cleaved caspase-3、Bax表达;双荧光素酶基因实验检测CircOGDH与miR-195-5p、PGAM5与miR-195-5p之间的靶向关系。结果:与NC组对比,OGD/R组miR-195-5p表达、OD450值均降低,CircOGDH、PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax蛋白表达均升高(P<0.05);对比OGD/R组和si-NC组,si-CircOGDH组miR-195-5p表达、OD450值均升高,CircOGDH、PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax蛋白表达均降低(P<0.05);miR-195-5p mimic组分别与OGD/R组、mimic NC组对比,CircOGDH无显著差异(P>0.05),miR-195-5p表达、OD450值均升高,PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax蛋白表达均降低(P<0.05);与si-CircOGDH+inhibitor NC组对比,si-CircOGDH+miR-195-5p inhibitor组CircOGDH差异不显著(P>0.05),miR-195-5p表达、OD450值均降低,PGAM5 mRNA表达、凋亡率、ROS及炎性因子水平、Cleaved caspase-3、Bax蛋白表达均升高(P<0.05)。双荧光素酶基因实验显示miR-195-5p和CircOGDH、PGAM5均存在靶向关系。结论:沉默CircOGDH可促进OGD/R诱导的HMC3细胞增殖,抑制其炎症反应和凋亡,可能与调控miR-195-5p/PGAM5轴有关。

基于UGM靶标的贵州药用植物抗结核分枝杆菌化合物构建及类似物研究

本文回顾性总结了潘卫东研究员项目团队有关二磷酸尿苷-半乳糖变异酶(UGM)探针设计、新型抑制剂的开发及新型筛选技术等方面的研究成果,明确了UGM反应机理,系统构建了贵州特色药用植物的抗结核分枝杆菌天然小分子化合物库,并从中发现了天然产物来源的UGM抑制剂,从而突破了当前UGM抑制剂成药性不佳的瓶颈,证实UGM存在变构位点,为贵州中药民族药的资源开发拓展了新思路。

Construction of Escherichia coli by Metabolic Engineering for Synthesis of Mesaconic Acid

Mesaconic acid has a special chemical structure and can undergo a series of reactions such as polymerization and addition. It is an important chemical intermediate and widely used in material, chemical and other industries. The chemical synthesis of mesaconic acid requires nitric acid, which is dangerous and harmful to the environment. The production of mesaconic acid by microbial fermentation has the characteristics of low raw material price, high efficiency and strong specificity, and thus a strong industrial application prospect. Mesaconic acid is an intermediate product of glutamic acid degradation pathway of microorganisms such as Clostridium tetani. However, at present, few reports have been conducted on the production of mesaconic acid by metabolic engineering microorganisms. In this study, glutamate mutase(GLM) and 3-methylaspartate ammonialyase(MAL) from C. tetani were recombined and expressed in Escherichia coli, and the obtained strain, BL21(DE3)/pETDuet-1-MAL-mutS-mutE, achieved the yield of mesaconic acid of 1.06 g/L. Compared with the wild type, the yields of mesaconic acid from mutants G133A and G133S increased by 21% and 16%, respectively. After 24 h of flask fermentation, the yields of mesaconic acid reached 1.28 and 1.23 g/L, respectively. This study can provide reference for microbial synthesis of mesaconic acid.

磷酸甘油酸变位酶1在结直肠癌组织中的表达及其对患者预后和癌细胞恶性生物学行为的影响

目的:探讨结直肠癌(CRC)组织中磷酸甘油酸变位酶1(PGAM1)的表达及其与患者预后的关系,研究PGAM1对CRC细胞增殖、迁移和侵袭的影响。方法:选择2003年3月至2008年11月间在天津医科大学肿瘤医院手术切除的30例CRC患者的肿瘤组织标本及临床资料,采用免疫组织化学染色法检测CRC组织中PGAM1蛋白的表达,分析PGAM1表达与患者临床病理特征的关系,Kaplan-Meier生存分析法比较PGAM1高表达与低表达患者的OS、PFS来评价PGAM1表达与患者预后的关系。利用RNA干扰技术分别将si-PGAM1及si-NC质粒转染至HCT-116和SW480细胞,WB法检测转染细胞中PGAM1蛋白的表达水平,CCK-8、Transwell实验分别检测敲低PGAM1对CRC细胞增殖、迁移和侵袭的影响。结果:30例CRC组织中PGAM1阳性染色定位于CRC细胞的细胞质,其中33.3%(10/30例)呈高表达。虽然PGAM1高表达与CRC患者年龄、性别、组织学类型、肿瘤大小、淋巴结转移、远处转移及临床TNM分期无关(均P>0.05),但是PGAM1高表达与低表达患者相比其OS、PFS显著缩短。在CRC细胞中敲低PGAM1后,细胞的增殖、迁移和侵袭能力均显著降低(均P<0.05)。结论:CRC组织中PGAM1呈高表达,PGAM1高表达的患者预后较差;敲低PGAM1后细胞的增殖、迁移及侵袭能力均显著降低,提示PGAM1可能是CRC患者预后的生物标志物。

镉胁迫对金银花生理生态特征的影响

采用水培试验方法,研究了不同浓度镉(Cd)(0、5、10、25和50mg·L-1)胁迫条件下藤本植物金银花的生长和生理特性.结果表明:与对照相比,Cd胁迫对金银花的生长未造成明显影响,在5～50mg·L-1Cd处理下,其生物量无明显差异(P>0.05),在低浓度Cd(5mg·L-1)处理下生物量有所增加,叶、根生物量和总生物量分别增加了2.88%、2.33%和1.25%,说明金银花对Cd具有较强的抗性.在低浓度Cd胁迫下,植物各器官的含水量和可溶性蛋白含量均有所降低,而根系和叶片的丙二醛含量分别增加51.90%和23.07%,叶绿素和类胡萝卜素含量则增加15.87%和24.89%,超氧化物歧化酶活性也显著增强.随着Cd浓度的增高,金银花体内的叶绿素和类胡萝卜素含量,以及超氧化物歧化酶活性均有所降低.

蒿甲醚对日本血吸虫磷酸葡萄糖变位酶、醛缩酶、磷酸甘油酸变位酶和烯醇化酶的影响

[目的 ]观察蒿甲醚 (Art)对小鼠体内日本血吸虫磷酸葡萄糖变位酶 (GPM )、醛缩酶 (ALD)、磷酸甘油酸变位酶 (PGM)和烯醇化酶 (ENO)的影响。 [方法 ]小鼠感染血吸虫尾蚴 4～ 5wk后 ,1次灌服Art10 0mg/kg或 30 0mg/kg ,并于 2 4～ 48h后剖杀 ,收集日本血吸虫雌虫和雄虫 ,按NADPH的生成量或NADH的耗用量测定虫体的上述 4种酶活力。 [结果 ]经Art 10 0mg/kg作用 2 4h后 ,雌虫的GPM、ALD、PGM和ENO活力分别较对照组下降 15 %、 19%、 5 0 %和 46 % ,其间差别均具有显著意义 ,而雄虫仅PGM和ENO活力分别下降 2 2 %和 32 % ;48h后 ,雄虫的GPM和ALD活力亦分别下降 2 1%和 18% ,雌虫的GPM、ALD、PGM和ENO及雄虫的PGM和ENO活力则进一步下降。经Art 30 0mg/kg作用后 2 4～ 48h ,雌虫和雄虫的上述 4种酶活力均明显下降 ,且呈一定的时间效应关系。 [结论 ]Art对日本血吸虫尤其是雌虫的上述 4种酶有抑制作用。

三角褐指藻磷酸甘油酸变位酶基因可能侧翼序列的筛选、克隆以及序列测定

采用RT PCR的方法从酵母中成功地得到了磷酸甘油酸变位酶的cDNA基因 ,分别用32 P和地高辛 ddUTP标记以用作探针。以32 P标记的探针筛选三角褐指藻基因组文库 ,获得了 4kb的阳性DNA片段 ;进一步分析发现 ,该 4kb片段的真正阳性区域是位于片段端部的30 6bp的序列 ,因此认为该序列为三角褐指藻磷酸甘油酸变位酶基因的侧翼部分。克隆该30 6bp的DNA片段 ,并且测定其序列。结果表明 ,该 30 6bp的DNA片段包含两个同向重复序列 ,每个重复序列的大小为 1 1 6bp ,在每个重复序列中均含有GGTTCAATGT区域 ,这与一般常见的真核基因 5′端的CAATbox有相似之处。

铅胁迫对桐花树幼苗根叶蛋白质及根抗氧化酶活性的影响

[目的]研究桐花树抗重金属铅(Pb2+)污染的特性。[方法]采用水培的方法,研究不同浓度(0.1、0.5、1.0、2.0、3.0 mmol/L)外源重金属铅(Pb2+)胁迫对桐花树幼苗根和叶蛋白质及根抗氧化酶活性的影响。[结果]桐花树幼苗在受胁迫过程中受到时间和铅浓度的双重影响。根和叶的蛋白质含量随着铅浓度的升高总体呈先增加后减少的趋势,随着胁迫时间的延长,根和叶的蛋白质含量总体也趋于减少,除胁迫15 d,根在1.0 mmol/L左右、叶在1.0～3.0 mmol/L高于胁迫3d的。随着铅浓度的升高,POD和SOD活性总体表现先升高后降低的趋势;随着胁迫时间的延长,低浓度组和对照组POD和SOD活性呈逐渐升高的趋势,高浓度组呈先升高后降低的趋势。[结论]桐花树对重金属铅污染有一定的抗性,但当浓度大于2.0 mmol/L时,桐花树生长受到抑制。

镉胁迫对凡纳滨对虾血清中一氧化氮合成酶和超氧化物歧化酶活性的影响

研究了不同浓度Cd2+(0.05、0.5和5mg.L-1)对凡纳滨对虾Litopeneaus vannamei血清中的总一氧化氮合成酶(NOS)、诱导型一氧化氮合成酶(iNOS)和超氧化物歧化酶(SOD)活性的影响。结果表明,镉胁迫促使总NOS活力升高依剂量而异,中(0.5mg.L-1)、低(0.05mg.L-1)剂量组凡纳滨对虾血清中总NOS活力在24—48h显著高于对照组,而高剂量组(5mg.L-1)在12—48h血清中总NOS活力与对照组相比无显著差别;iNOS活力变化趋势与总NOS活力相似,但中、低剂量组之间iNOS活性无显著差异。镉胁迫促使凡纳滨对虾血清中SOD活力短暂升高,但高剂量在24—48h对SOD活力呈明显的抑制效应。

力竭性运动时大鼠脑组织自由基产生及氧化、抗氧化能力的动态观察

以大鼠递增负荷力竭性运动为模型，利用低温电子自旋共振（ＥＳＲ）技术，分别测定安静时、运动过程中、运动后即刻、运动后恢复期３０ｍｉｎ、２ｈ、４ｈ及８ｈ脑组织的氧自由基（ＯＴＲ）信号强度，同时测定ＳＯＤ活力和ＭＤＡ含量。结果显示：脑组织在运动过程中ＯＦＲ信号强度逐渐增加，并具有运动强度依赖的阶段性，恢复期４小时达到高峰；ＳＯＤ活性运动时无明显改变，恢复期２ｈ升至峰值；脂质过氧化水平（ＭＤＡ）在整个运动过程中及恢复期无显著变化。提示该运动模型所引起的脂质过氧化尚未累及中枢神经系统，并且脑组织可能存在重要而有效的抗氧化体系。

PGAM1通过激活Warburg效应调控宫颈癌细胞恶性生物学行为的机制研究

目的探讨磷酸甘油酸变位酶1(PGAM1)在宫颈癌组织中的表达情况以及可能的生物学机制。方法收集行手术切除的67例宫颈癌患者的新鲜宫颈癌组织及癌旁正常组织,采用免疫组化法检测PGAM1的表达情况并分析与患者病理特征的关系。构建PGAM1基因沉默稳转Hela宫颈癌细胞株,分析PGAM1基因沉默对Hela细胞凋亡和增殖活性的影响。采用qRT-PCR和Western blot法检测PGAM1对Akt/m TOR信号通路关键分子的影响。结果 (1)宫颈癌组织中PGAM1高表达率为58.21%,明显高于癌旁组织(P<0.05)。(2)不同临床分期、分化程度、浸润深度的患者宫颈癌组织中PGAM1的高表达率差异有统计学意义(P<0.05)。(3)Hela细胞sh PGAM1基因沉默后,细胞凋亡率较空白对照组(NC组)明显增加,细胞增殖活性明显降低(P<0.05)。(4)Hela细胞sh PGAM1基因沉默后,与NC组比较,PTEN mRNA和蛋白相对表达量明显上调,而p-Akt、p-m TOR mRNA和蛋白相对表达量明显降低(P<0.05)。结论肿瘤组织PGAM1蛋白高表达与宫颈癌的发生、发展有关,通过激活Akt/m TOR信号通路诱导的Warburg效应,促使肿瘤细胞的增殖,抑制其凋亡。

刚地弓形虫磷酸甘油酸变位酶2基因片段克隆、表达及抗原性分析

目的克隆、表达刚地弓形虫(Toxoplasma gondii)磷酸甘油酸变位酶2(TgPGAM2)基因片段,并分析其抗原性。方法提取弓形虫RH株速殖子总RNA,逆转录合成cDNA。PCR扩增TgPGAM2基因。扩增产物经双酶切后连接入pET30a(+)载体,重组质粒转化大肠埃希菌(E.coli)DH5α,阳性菌落经PCR和双酶切鉴定,并测序。将测序正确的重组质粒pET30a(+)-TgPGAM2转化至E.coli BL21并加入异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结合考马斯亮蓝染色检测表达产物。以兔抗弓形虫血清为一抗,蛋白质印迹(Western blotting)分析重组蛋白的抗原性。结果PCR扩增产物约为750 bp。菌落PCR、双酶切和测序结果显示,重组质粒pET30a(+)-TgPGAM2构建成功。SDS-PAGE结果显示,经IPTG诱导获得相对分子质量(Mr)约30 000的可溶性重组蛋白。Western blotting分析证实其能被兔抗弓形虫血清识别。结论刚地弓形虫RH株TgPGAM2基因片段可在原核表达系统中表达,且该可溶性重组蛋白具有抗原性。

SEQUENCE ANALYSIS OF A NOVEL INSECT PHOSPHOGLYCERATEMUTASE GENE FROM THE CHINESE HONEYBEE, APIS CERANA

A clone inserted with 1 104 bp fragment containing a 765bp Open Reading Frame(ORF), encoding a putative 2,3-bisphosphoglycerate(2,3BPG) dependent Phosphoglycerate mutase(dPGAM) that catalyzes the transfer of a phosphate group from the C3 carbon atom to the C2 carbon atom of phosphoglycerate, was screened by mass sequencing from the cDNA library of the venom glands of Apis cerana. The deduced amino acid sequence shared high similarities (39%-88%)with the dPGAM of 7 other organisms, but the similarities with the iPGAM of 4 other organisms were low (10%-12%). Moreover, the alignment of Ac-PGAM with the dPGAMs from 7 other organisms showed that all the active site amino acid residues were conserved. This result shows that Ac-PGAM is a typical dPGAM. Thus, this is the second PGAM gene reported in Insecta. Furthermore, phylogenetic analysis showed that the evolutionary tree of PGAMs reflects the systematic relationship of species.